NH₂OH Disproportionation Mediated by Anaerobic Ammonium-oxidizing (Anammox) Bacteria

Abstract

Anammox bacteria produce N₂ gas by oxidizing NH₄⁺ with NO₂^–, and hydroxylamine (NH₂OH) is a potential intermediate of the anammox process. N₂ gas production occurs when anammox bacteria are incubated with NH₂OH only, indicating their capacity for NH₂OH disproportionation with NH₂OH serving as both the electron donor and acceptor. Limited information is currently available on NH₂OH disproportionation by anammox bacteria; therefore, the stoichiometry of anammox bacterial NH₂OH disproportionation was examined in the present study using ¹⁵N-tracing techniques. The anammox bacteria, Brocadia sinica, Jettenia caeni, and Scalindua sp. were incubated with the addition of ¹⁵NH₂OH, and the production of ¹⁵N-labeled nitrogenous compounds was assessed. The anammox bacteria tested performed NH₂OH disproportionation and produced ^15-15N₂ gas and NH₄⁺ as reaction products. The addition of acetylene, an inhibitor of the anammox process, reduced the activity of NH₂OH disproportionation, but not completely. The growth of B. sinica by NH₂OH disproportionation (–240.3‍ ‍kJ mol NH₂OH^–1 under standard conditions) was also tested in 3 up-flow column anammox reactors fed with 1) 0.7‍ ‍mM NH₂OH only, 2) 0.7‍ ‍mM NH₂OH and 0.5‍ ‍mM NH₄⁺, and 3) 0.7‍ ‍mM NH₂OH and 0.5‍ ‍mM NO₂^–. NH₂OH consumption activities were markedly reduced after 7‍ ‍d of operation, indicating that B. sinica was unable to maintain its activity or biomass by NH₂OH disproportionation.

Anaerobic ammonium-oxidizing (anammox) bacteria were initially discovered in a denitrifying bioreactor in 1995 (Mulder et al., 1995), and are now recognized as the main players in the global nitrogen cycle (Kuypers et al., 2003; 2005; Amano et al., 2007; Yoshinaga et al., 2011). In the anammox process, NH₄⁺ is oxidized to N₂ gas using NO₂^– as an electron acceptor, and the stoichiometry of the anammox process has been described as follows (Lotti et al., 2014):

1 NH₄⁺+1.146 NO₂^–+0.071 HCO₃^–+0.057 H⁺

→ 0.986 N₂+0.161 NO₃^–+0.071 CH_1.74O_0.31N_0.20+2.002 H₂O eq. 1

As an intermediate, hydrazine (N₂H₄) is synthesized from NH₄⁺ and NO or NH₂OH by hydrazine synthase, and its biosynthesis appears to be unique to metabolism by anammox bacteria (Strous et al., 2006; Kartal et al., 2011; Oshiki et al., 2016a). Anammox bacteria are monophyletically affiliated into the bacterial order Brocadiales in the phylum Planctomycetota (Strous et al., 1999), and five candidate genera have been proposed: Candidatus Brocadia, Kuenenia, Jettenia, Anammoxoglobus, and Scalindua (Egli et al., 2003; Kuypers et al., 2003; Quan et al., 2008). These anammox bacteria have been detected in various natural and man-made ecosystems and are significantly involved in nitrogen loss (Oshiki et al., 2016b).

Hydroxylamine (NH₂OH), a well-known intermediate of the aerobic NH₃ oxidation reaction (Madigan et al., 2019), is also a potential intermediate of the anammox process (van de Graaf et al., 1997). The anammox bacterium, Brocadia sinica reduced NO₂^– to NH₂OH (Oshiki et al., 2016a) potentially by using reductive hydroxylamine dehydrogenase (rHao) (Ferousi et al., 2021), and synthesized N₂H₄ from the NH₂OH and NH₄⁺ using hydrazine synthase (Oshiki et al., 2016a). Kuenenia stuttgartiensis reduced NO₂^– to NO (Kartal et al., 2011), and NO was further reduced to NH₂OH by hydrazine synthase to synthesize N₂H₄ (Dietl et al., 2015). Apart from the anammox process, the nitrogen transformation reaction, NH₂OH disproportionation, has been reported in anammox bacteria (van der Star et al., 2008). A disproportionation reaction is a reaction in which a chemical compound serves as both an electron donor and accepter, and the disproportionation reaction of inorganic sulfur (Finster, 2008) is an example of a microbial disproportionation reaction. In NH₂OH disproportionation, NH₂OH is converted to N₂ gas and NH₄⁺ using the following stoichiometry (Pacheco et al., 2011):

3 NH₂OH+H⁺ → NH₄⁺+N₂+3 H₂O eq. 2

NH₂OH disproportionation involves the following 2 reactions, N₂H₄ production and consumption (Soler-Jofra et al., 2020):

NH₄⁺+NH₂OH → N₂H₄+H₂O+H⁺ eq. 3

2 NH₂OH+N₂H₄+2 H⁺ → 2 NH₄⁺+N₂+2 H₂O eq. 4

Although NH₂OH disproportionation by K. stuttgartiensis (van der Star et al., 2008; Soler-Jofra et al., 2020) and B. sinica (Oshiki et al., 2016a) has been described, further studies are required to obtain a more detailed understanding of anammox bacterial NH₂OH disproportionation for the following reasons. Although the kinetics of NH₂OH disproportionation have been investigated using K. stuttgartiensis (van der Star et al., 2008; Soler-Jofra et al., 2020), the amounts of N₂ gas produced in NH₂OH disproportionation (see eq. 2) were not measured in previous studies and the stoichiometry of NH₂OH disproportionation was not established. Furthermore, we previously examined NH₂OH disproportionation by B. sinica; however, we only reported the occurrence of NH₂OH disproportionation (Oshiki et al., 2016a) and did not investigate the effects of NH₂OH concentrations on NH₂OH disproportionation by repeating batch incubations. In addition, although eq. 2 yields –‍240.3‍ ‍kJ mol NH₂OH^–1 of free energy under standard conditions (Soler-Jofra et al., 2020), the growth of anammox bacteria with NH₂OH disproportionation has not yet been examined.

Therefore, the present study investigated anammox bacterial NH₂OH disproportionation. The phylogenetically different anammox bacteria, B. sinica, Jettenia caeni, and Scalindua sp. were incubated with ¹⁵N-labeled NH₂OH, and the stoichiometry of NH₂OH disproportionation was carefully assessed based on measurements of ^15-15N₂ and NH₄⁺ concentrations. NH₂OH disproportionation was also analyzed under acetylene inhibition conditions. Acetylene is a strong inhibitor of aerobic NH₃ oxidation, N₂O reduction to N₂ (i.e., denitrification), and the anammox process (Jensen et al., 2007); however, its effects on NH₂OH disproportionation currently remain unknown. B. sinica was cultured in up-flow column reactors with the addition of NH₂OH to establish whether it grows on NH₂OH, and the activity of NH₂OH consumption and the abundance of the anammox bacterial 16S rRNA gene were evaluated.

Materials and Methods

Anammox bacterial cultures

Planktonic cells of B. sinica, J. caeni, and Scalindua sp. were cultivated in membrane bioreactors (MBRs) equipped with a hollow fiber membrane module (pore size of 0.1‍ ‍μm, polyethylene) as previously described (Oshiki et al., 2013; Zhang and Okabe, 2020). Culture media fed into MBRs contained KH₂PO₄ (24.4‍ ‍mg L^–1), MgSO₄·7H₂O (60‍ ‍mg L^–1), CaCl₂ (51‍ ‍mg L^–1), yeast extract (Becton, Dickinson and Company) (1.0‍ ‍mg L^–1), and 0.5‍ ‍mL of trace element solutions I and II (van de Graaf et al., 1996). The artificial sea salt SEALIFE (Marine Tech) (Kindaichi et al., 2011) was supplemented into media for “Ca. Scalindua sp.” at a final concentration of 28‍ ‍g L^–1. Equimolar amounts of NH₄(SO₄)₂ and NaNO₂ were supplemented into media at 10‍ ‍mM for B. sinica and Scalindua sp. and 5‍ ‍mM for J. caeni, and the nitrogen loading rates of the MBRs for B. sinica, J. caeni, and Scalindua sp. were set at 0.55, 0.18, and 0.45‍ ‍kg N m^–3 d^–1, respectively. MBRs were operated at 37°C for B. sinica and at 25°C for J. caeni and Scalindua sp. pH was not controlled in MBRs, but was in the range of pH 7.6–8.0. Anammox bacterial cells accounted for more than 90% of the total biomass in MBRs as measured by a fluorescence in-situ hybridization (FISH) ana­lysis using the oligonucleotide probes AMX820 (Schmid et al., 2001) and EUBmix composed of equimolar EUB338, EUB338II, and EUB338III (Daims et al., 1999). Anammox bacterial species were routinely checked based on the partial anammox bacterial 16S rRNA gene sequence using Sanger sequencing (Oshiki et al., 2011).

Batch incubations of anammox bacteria

Standard anaerobic techniques were employed in an anaerobic chamber (Coy Laboratories Products) in which the concentration of oxygen was maintained at <1 ppm. Culture media and stock solutions were prepared by purging N₂ gas for >30‍ ‍min, and then repeatedly vacuuming and purging He gas. The ¹⁵N enrichment of ¹⁵NH₂OH·HCl (Cambridge Isotope Laboratories) was >98%.

Anammox bacterial cells collected from MBRs were centrifuged at 13,420×g at 20°C for 10‍ ‍min, washed, and then resuspended in the above culture media without NH₄⁺ and NO₂^– at concentrations of 0.5‍ ‍mg protein mL^–1. Twenty-five milliliters of the cell suspension was dispensed into 70-mL serum glass vials (Nichiden-Rika glass), and the headspace was replaced with He gas (>99.99995%) after sealing with butyl rubber stoppers and aluminium caps. Vials were incubated after the addition of ¹⁵NH₂OH (final concentration of 1.0 to 10‍ ‍mM) and acetylene (30‍ ‍μM) (Jensen et al., 2007) in the dark at 37°C for B. sinica and at 25°C for J. caeni and Scalindua sp.. Liquid samples were collected using a 1-mL plastic disposable syringe, immediately filtered using a 0.2-μm cellulose acetate filter, and subjected to measurements of NH₄⁺, NO₂^–, NO₃^–, and NH₂OH concentrations. Gas samples were collected using a gas-tight glass syringe and immediately injected into a gas chromatograph to assess ^14-15N₂ and ^15-15N₂ concentrations.

Up-flow column reactors fed with NH₂OH

Three 255-mL up-flow column reactors were operated at 37°C in the dark with the continuous feeding of the above culture media containing 1) 0.7‍ ‍mM ¹⁴NH₂OH, 2) 0.7‍ ‍mM ¹⁴NH₂OH and 0.5‍ ‍mM ¹⁴NH₄⁺, or 3) 0.7‍ ‍mM ¹⁴NH₂OH and 0.5‍ ‍mM ¹⁴NO₂^–. B. sinica cells immobilized on polyvinyl alcohol (PVA)-sodium alginate (SA) (6 and 2% [w/v], respectively) beads were inoculated into the column reactors at a packing ratio of 50% (v/v). The gel immobilization of B. sinica cells in PVA-SA gel beads was performed as previously described (Ali et al., 2015). Briefly, the planktonic cells of B. sinica collected from the above MBR were resuspended in culture media, and mixed with an equal volume of PVA-alginate (FUJIFILM Wako) solution (12 and 4% [w/v], respectively). The gel solution was dropped using a disposable 50-mL plastic syringe (Terumo) into a 4% (w/v) CaCl₂ solution to form gel beads (diameter of ca. 2‍ ‍mm). After an overnight incubation at 20°C, gel beads were washed with fresh inorganic medium. The gel beads obtained were inoculated into the up-flow column reactors.

Chemical ana­lysis

NH₄⁺, NO₂^–, and NO₃^– concentrations were measured using the ion chromatograph IC-2010 equipped with the TSKgel SuperIC-Anion HS or TSKgel SuperIC-Cation HS column (Tosoh). NH₂OH concentrations were measured colorimetrically (Frear and Burrell, 1955). Briefly, liquid samples were mixed with 0.48% (w/v) trichloroacetic acid, 0.2% (w/v) 8-hydroxyquinoline, and 0.2 M Na₂CO₃, heated at 100°C for 1‍ ‍min, and absorbance was then measured at a wavelength of 705‍ ‍nm using the spectrophotometer V-630bio (Jasco). N₂H₄ concentrations were measured colorimetrically using p-dimethyl-aminobenzaldehyde (Watt and Chrisp, 1952). Briefly, liquid samples were mixed with 0.12 M p-dimethyl-aminobenzaldehyde, and absorbance was measured at a wavelength of 460‍ ‍nm.

^14-15N₂ and ^15-15N₂ concentrations were measured by gas chromatography mass spectrometry (GC/MS) (Isobe et al., 2011a; 2011b). Fifty microliters of the headspace gas was collected using a 100-‍μL gas-tight glass syringe and immediately injected into the gas chromatograph GCMS-QP 2010 SE (Shimadzu) equipped with a fused silica capillary column (Agilent Technologies). Peaks at m/z=29 and 30 corresponding to ^14-15N₂ and ^15-15N₂ were monitored, and concentrations were calculated using a standard curve prepared using ^15-15N₂ gas (Cambridge Isotope Laboratories). The ^14-15N₂ and ^15-15N₂ concentrations of ¹⁵NH₂OH were calculated by considering the ¹⁵N enrichment of ¹⁵NH₂OH·HCl (¹⁵N, 98%) and the natural abundance of ^14-15N₂ and ^15-15N₂ in atmospheric N₂ gas contaminated at the injection of the gas sample.

qPCR assay

The copy numbers of the anammox bacterial 16S rRNA gene were measured using a qPCR assay. Genomic DNA was extracted from gel beads collected from the up-flow column reactors using the FastDNA SPIN kit (Qiagen) according to the instruction manual supplied by the manufacturer. The qPCR assay was conducted using the ABI7500 fast Real-Time PCR System (Thermo Fisher Scientific) and Premix Ex Taq (Probe qPCR) (TakaraBio) under previously described thermal conditions (Zhang and Okabe, 2017a). The oligonucleotide primers and TaqMan probe used for the PCR amplification of the B. sinica 16S rRNA gene were BRS95F, BRS170R, and BRS130P. Standard curves (10¹ to 10⁶ copies μL^–1) were prepared using a dilution series of plasmid DNAs containing a partial B. sinica 16S rRNA gene sequence.

Results

NH₂OH disproportionation by phylogenetically different anammox bacteria

Anammox bacterial cells of B. sinica, J. caeni, and Scalindua sp. were incubated with the addition of 1.0 to 2.5‍ ‍mM ¹⁵NH₂OH. As shown in Fig. 1a, c, and e, ¹⁵NH₂OH consumption occurred in the culture concurrently with the production of ^15-15N₂ gas and NH₄⁺. N₂H₄ was also produced and markedly increased when NH₂OH concentrations decreased below 0.1‍ ‍mM (i.e., a 60-min incubation for B. sinica and Scalindua sp. and a 120-min incubation for J. caeni). ^14-15N₂, NO₂^–, and NO₃^– were not detected during any incubations, and ¹⁵NH₂OH consumption was negligible in vials without anammox bacterial cells (i.e., abiotic control incubation).

Fig. 1.

¹⁵NH₂OH-tracing batch incubations to demonstrate anammox bacterial NH₂OH disproportionation. Twenty-five milliliters of Brocadia sinica (panel a and b), Jettenia caeni (panel c and d), and Scalindua sp. (panel e and f) cultures were incubated with the addition of 2.5‍ ‍mM ¹⁵NH₂OH in 70-mL glass vials. Incubations were repeated with the addition of 30‍ ‍μM acetylene (panel b, d, and f). All incubations were performed in triplicate, and symbols and error bars represent mean values and the range of standard deviations, respectively. The standard deviations of data points are often within the symbols.

Batch incubations were repeated with the addition of 30‍ ‍μM acetylene. Anammox bacterial cells consumed ¹⁵NH₂OH (Fig. 1b, d, and f), whereas consumption rates were >3-fold lower than those without acetylene. Although ^15-15N₂ gas and NH₄⁺ were produced during the incubation, N₂H₄ was not produced in any anammox bacterial cultures.

The above batch incubation of B. sinica was repeated with an increase in the initial NH₂OH concentration (from 5 to 10‍ ‍mM) with/without 30‍ ‍μM acetylene. As shown in Fig. 2, ¹⁵NH₂OH consumption and the concomitant production of ^15-15N₂ and NH₄⁺ occurred, similar to the batch incubation with the addition of 2.5‍ ‍mM ¹⁵NH₂OH (i.e., Fig. 1a and b). It is important to note that N₂H₄ production only occurred when NH₂OH concentrations decreased below 1.5‍ ‍mM (Fig. 2a after 7.5 h of the incubation), and N₂H₄ production was not observed in Fig. 2c and e.

Fig. 2.

Effects of the initial NH₂OH concentration on NH₂OH disproportionation by Brocadia sinica. Twenty-five milliliters of the Brocadia sinica culture was incubated with the addition of 5‍ ‍mM (panel a and b), 7.5‍ ‍mM (panel c and d), and 10‍ ‍mM ¹⁵NH₂OH (panel e and f) in 70-mL glass vials. Incubations were repeated with the addition of 30‍ ‍μM acetylene (panel b, d, and f). All incubations were performed in triplicate, and symbols and error bars represent mean values and the range of standard deviations, respectively. The standard deviations of data points are often within the symbols.

The stoichiometry and nitrogen mass balance of the above batch incubations are shown in Table 1. The theoretical values for Δ^15-15N₂/Δ¹⁵NH₂OH and Δ¹⁵NH₄⁺/Δ¹⁵NH₂OH were 0.33 and 0.33, respectively when NH₂OH disproportionation occurred according to eq. 2. The values for Δ^15-15N₂/Δ¹⁵NH₂OH and Δ¹⁵NH₄⁺/Δ¹⁵NH₂OH obtained from batch incubations with the addition of 2.5 or 5‍ ‍mM ¹⁵NH₂OH were in the range of 0.19–0.45 and 0.17–0.36, respectively. These values were generally close to the theoretical values and those for Δ¹⁵NH₄⁺/Δ¹⁵NH₂OH in the K. stuttgartiensis culture (0.19–0.48), except for the following batch incubations: B. sinica with 2.5‍ ‍mM ¹⁵NH₂OH without acetylene (0.2 for Δ¹⁵NH₄⁺/Δ¹⁵NH₂OH), B. sinica with 5‍ ‍mM ¹⁵NH₂OH with acetylene (0.19 for Δ^15-15N₂/Δ¹⁵NH₂OH), and Scalindua sp. with 2.5‍ ‍mM ¹⁵NH₂OH with and without acetylene (0.17 or 0.21 for Δ¹⁵NH₄⁺/Δ¹⁵NH₂OH). Batch incubations of B. sinica with 7.5 or 10‍ ‍mM NH₂OH generally resulted in lower values for Δ^15-15N₂/Δ¹⁵NH₂OH and Δ¹⁵NH₄⁺/Δ¹⁵NH₂OH, which were in the ranges of 0.14–0.19 and 0.09–0.19, respectively.

Table 1. Stoichiometry of NH₂OH disproportionation mediated by anammox bacteria. The values obtained in the present study were mean values from triplicate biological replicates, and values were calculated from the initial and final concentrations during batch incubations. The N-mass balance (%) was calculated by dividing the total amounts of ¹⁵NH₂OH, ¹⁵NH₄⁺, and ^15-15N₂ at the end of the incubation (μmol [N vial]^–1) by the initial amounts of ¹⁵NH₂OH (μmol [N vial]^–1).

Species	NH2OH (mM)	Acetylene*	N-mass balance	Δ15-15N2/ΔNH2OH (mol/mol)	Δ15NH4+/ΔNH2OH (mol/mol)	References
B. sinica	2.5	w/o	91%	0.35	0.20	This study
	5	w/o	85%	0.28	0.27
	7.5	w/o	66%	0.19	0.12
	10	w/o	72%	0.16	0.09
	2	+	91%	0.27	0.35
	5	+	81%	0.19	0.30
	7.5	+	76%	0.16	0.19
	10	+	77%	0.14	0.15
J. caeni	1	w/o	83%	0.26	0.25
		+	91%	0.26	0.36
Scalindua sp.	2	w/o	82%	0.29	0.17
	2.5	+	111%	0.45	0.21
K. stuttgartiensis	1.6–10	w/o	NA	ND	0.19–0.48	van der Star et al., 2008 Soler-Jofra et al., 2020
B. flugida	4	w/o	NA	ND	0.25	van der Star et al., 2008

*; w/o and +: without and with the addition of 30‍ ‍μM acetylene, respectively, NA: not available, ND: not determined.

Cultivation of B. sinica with NH₂OH in up-flow column reactors

NH₂OH consumption and the abundance of the anammox bacterial 16S rRNA gene were examined in the 1) NH₂OH-, 2) NH₂OH and NH₄⁺-, and 3) NH₂OH and NO₂^–-feeding up-flow column reactors. In all operated reactors, NH₂OH consumption markedly decreased after 7‍ ‍d of operation, and halted after 15‍ ‍d of operation (Fig. 3). NH₄⁺ concentrations in the effluents of the NH₂OH- and NH₂OH and NO₂^–-feeding reactors were in the same range (Fig. 3a and c, respectively), and the consumption of NH₄⁺ did not occur in the NH₂OH and NH₄⁺-feeding reactor (Fig. 3b). The copy numbers of the anammox bacterial 16S rRNA gene decreased over time, and its abundance after 14‍ ‍d of operation was an order of magnitude less than that after 1‍ ‍d of operation. Copy numbers were not measured in the reactor fed with NH₂OH and NO₂^– (Fig. 3c).

Fig. 3.

NH₂OH consumption by Brocadia sinica in continuous up-flow column reactors. Up-flow column reactors were inoculated with PVA-SA gel beads immobilizing B. sinica cells and were operated with the continuous feeding of NH₂OH (panel a), NH₂OH and NH₄⁺ (panel b), and NH₂OH and NO₂^– (panel c).

Discussion

NH₂OH disproportionation was examined using phylogenetically different anammox bacteria, and the present study clearly indicated that NH₂OH disproportionation is a common nitrogen transformation process of anammox bacteria (Table 1). Although previous studies (van der Star et al., 2008; Soler-Jofra et al., 2020) reported anammox bacterial NH₂OH disproportionation, they did not examine the amount of N₂ gas produced. The present study performed sophisticated ¹⁵NH₂OH-tracing batch incubations, and revealed that anammox bacteria yielded both N₂ gas and NH₄⁺ from NH₂OH disproportionation. ¹⁵NH₂OH-tracing batch incubations enabled the stoichiometry of ^15-15N₂ and ¹⁵NH₄⁺ production to ¹⁵NH₂OH consumption to be examined (Table 1), and the results obtained indicated that an increase in the initial concentration of NH₂OH resulted in a decrease in Δ^15-15N₂/Δ¹⁵NH₂OH and Δ¹⁵NH₄⁺/Δ¹⁵NH₂OH for B. sinica. Furthermore, the addition of acetylene resulted in a decrease and increase in Δ^15-15N₂/Δ¹⁵NH₂OH and Δ¹⁵NH₄/Δ¹⁵NH₂OH, respectively. These variations in stoichiometry suggested that ¹⁵N₂ and/or ¹⁵NH₄⁺ were produced by multiple nitrogen transformation pathways other than NH₂OH disproportionation. Anammox bacterial hydroxylamine dehydrogenase oxidizes NH₂OH to NO (Maalcke et al., 2014), and N₂ gas may be produced using the NO formed by the anammox process (the coupling of NH₄⁺ and NO) (Kartal et al., 2013). Although the reduction of NH₂OH to NH₄⁺ by anammox bacterial cells has not yet been demonstrated, anammox bacteria are capable of dissimilatory nitrite reduction to ammonium (Kartal et al., 2007).

N₂ and NH₄⁺-forming NH₂OH disproportionation has not yet been reported for microorganisms other than anammox bacteria. It requires the formation of a nitrogen-nitrogen bond for N₂ gas, and only nitric oxide reductase and hydrazine synthase catalyze this reaction (Dietl et al., 2015). This may be the reason why N₂ and NH₄⁺-forming NH₂OH disproportionation has not yet been detected in microbial cultures other than anammox bacteria. Apart from N₂ and NH₄⁺-forming NH₂OH disproportionation, 1) NH₄⁺ and NO-, 2) NH₄⁺ and NO₂^–-, and 3) N₂O and NH₄⁺-forming NH₂OH disproportionation are thermodynamically favorable (Pacheco et al., 2011); however, limited information is currently available on their activities or involvement in the nitrogen transformation process in natural and man-made ecosystems.

The addition of acetylene did not induce the accumulation of N₂H₄, but reduced NH₂OH consumption (Fig. 1b, d, and f), which indicated that acetylene inhibited the enzymes involved in both NH₂OH consumption and N₂H₄ production reactions. B. sinica hydrazine synthase utilizes NH₂OH as a substrate for N₂H₄ synthesis (Oshiki et al., 2016a); therefore, hydrazine synthase may be the enzyme that is inhibited by acetylene. The inhibition of anammox bacterial activity by acetylene has been reported (Jensen et al., 2007); however, the underlying mechanism(s) remain unknown. Apart from hydrazine synthase, acetylene inhibited the NO₂^– reduction reaction of anammox bacteria (Kartal et al., 2011; Oshiki et al., 2016a), indicating that acetylene suppresses multiple nitrogen transformation reactions of anammox bacteria. Although the acetylene inhibition of copper-containing metalloproteins, such as ammonia monooxygenase and nitrous oxide reductase (Amo and Nos, respectively) has been described (Ensign et al., 1993; Gilch et al., 2009), the involvement of copper-containing metalloproteins in anammox bacterial metabolism, particularly N₂H₄ production, remains unclear. The binding site of acetylene to anammox bacterial enzymes is of interest for obtaining a more detailed understanding of the acetylene inhibition of anammox bacteria. The addition of 30‍ ‍μM acetylene did not completely inhibit the production of ^15-15N₂ gas or NH₄⁺ (Fig. 1 and 2). This result suggests the incomplete inhibition of anammox bacterial hydrazine synthase with the addition of 30‍ ‍μM acetylene (Jensen et al., 2007) and/or the production of ^15-15N₂ gas and NH₄⁺, but not through N₂H₄. The corresponding mechanism has not yet been elucidated, and a further understanding of acetylene inhibition by anammox bacteria will provide novel insights.

Apart from the above acetylene inhibition, high NH₂OH concentrations (>0.1‍ ‍mM NH₂OH in Fig 1a, c, and e, and >1.5‍ ‍mM NH₂OH in Fig. 2a) did not induce the accumulation of N₂H₄. This behavior cannot simply be explained by the inhibition of anammox bacterial hydrazine dehydrogenase involved in N₂H₄ oxidation to N₂. The N₂H₄ oxidation activities of purified anammox bacterial hydrazine dehydrogenase were inhibited in the presence of 2.4 to 7.9‍ ‍μM NH₂OH (Shimamura et al., 2007; Maalcke et al., 2016), and this inhibition provides an explanation for the accumulation of N₂H₄ during batch incubations, but not for the lack of its accumulation at high NH₂OH concentrations. On the other hand, anammox bacterial N₂H₄ accumulation may be explained by the balance between N₂H₄ production and consumption reactions (eq. 3 and 4, respectively), as reported in previous studies (van der Star et al., 2008; Soler-Jofra et al., 2020). N₂H₄ production and consumption reactions require 1 and 2 moles of NH₂OH, respectively, and higher NH₂OH concentrations increase N₂H₄ consumption rates more than N₂H₄ production rates; therefore, N₂H₄ does not accumulate at high NH₂OH concentrations. The affinity constants of eq. 3 and 4 for NH₂OH need to be examined in more detail in order to clarify the N₂H₄ accumulation behavior of anammox bacterial cells.

The results of the up-flow column reactor experiments revealed that B. sinica did not proliferate with NH₂OH disproportion (Fig. 3), and this is the first experimental evidence to show that an anammox culture cannot be maintained with NH₂OH as the sole energy source. A 1 log reduction in the B. sinica 16S rRNA gene copy number clearly indicated that B. sinica did not proliferate in the operated reactors. B. sinica cells may have been compromised during reactor operation due to the high toxicity and mutagenesis of NH₂OH, which resulted in a decrease in the B. sinica 16S rRNA gene copy number in PVA-SA gel beads. We previously reported a log reduction in the anammox bacterial 16S rRNA gene copy number in PVA-SA gel beads under unfavorable cultivation conditions (Zhang et al., 2017b). It is important to note that B. sinica cells preferentially performed NH₂OH disproportionation over anammox. Anammox using NO₂^– or NH₂OH did not occur in the NH₂OH and NO₂^–- or NH₂OH and NH₄⁺-feeding reactors (Fig. 3c and b, respectively), which indicated that the anammox activities of B. sinica were inhibited in these reactors by NH₂OH. Therefore, low NH₂OH concentrations need to be maintained in order to avoid the inhibition of anammox activities and achieve the stable performance of nitrogen removal. Although the inhibitory effects of NH₂OH on nitrifying bacteria have been investigated (Kindaichi et al., 2004; Soler-Jofra et al., 2021), the IC₅₀ concentration of NH₂OH for anammox bacteria has not yet been systematically examined. In the present study, the sudden deterioration of NH₂OH consumption occurred after 7‍ ‍d of operation; however, the concentration of NH₂OH in the influent was markedly lower than those in batch incubations in which the marked deterioration of NH₂OH consumption did not occur (i.e., 0.7 and 2.5–10‍ ‍mM, respectively). Therefore, it was not possible to approximate the IC₅₀ concentration of NH₂OH inhibition from short-term batch incubations, and further studies are warranted to assess the IC₅₀ concentration of the long-term inhibition of NH₂OH. NH₂OH may accumulate at the submicromolar range in natural aquatic environments (Fukumori et al., 2003; Bikbulatova et al., 2007) and at the submillimolar range in nitrifying cultures (Soler-Jofra et al., 2021 and references therein); however, the impact of NH₂OH on nitrogen transformation reactions remains unclear. Further studies are needed to examine NH₂OH transformation, including anammox bacterial NH₂OH disproportionation, in natural and man-made ecosystems.

Citation

Oshiki, M., Gao, L., Zhang, L., and Okabe, S. (2022) NH₂OH Disproportionation Mediated by Anaerobic Ammonium-oxidizing (Anammox) Bacteria. Microbes Environ 37: ME21092.

https://doi.org/10.1264/jsme2.ME21092

Acknowledgements

This work was supported by JSPS KAKENHI Grant numbers 17K15305 and 19K05805 to M.O. and 19H00776 to S.O., respectively. The authors declare no conflicts of interest associated with this manuscript.

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